ZNF91 deletion in human embryonic stem cells leads to ectopic activation of SVA retrotransposons and up-regulation of KRAB zinc finger gene clusters.

Transposable element (TE) invasions have shaped vertebrate genomes over the course of evolution. They have contributed an extra layer of species-specific gene regulation by providing novel transcription factor binding sites. In humans, SINE-VNTR-Alu (SVA) elements are one of three still active TE families; approximately 2800 SVA insertions exist in the human genome, half of which are human-specific. TEs are often silenced by KRAB zinc finger (KZNF) proteins recruiting corepressor proteins that establish a repressive chromatin state. A number of KZNFs have been reported to bind SVAs, but their individual contribution to repressing SVAs and their roles in suppressing SVA-mediated gene-regulatory effects remains elusive. We analyzed the genome-wide binding profile for ZNF91 in human cells and found that ZNF91 interacts with the VNTR region of SVAs. Through CRISPR-Cas9-mediated deletion of ZNF91 in human embryonic stem cells, we established that loss of ZNF91 results in increased transcriptional activity of SVAs. In contrast, SVA activation was not observed upon genetic deletion of the ZNF611 gene encoding another strong SVA interactor. Epigenetic profiling confirmed the loss of SVA repression in the absence of ZNF91 and revealed that mainly evolutionary young SVAs gain gene activation-associated epigenetic modifications. Genes close to activated SVAs showed a mild up-regulation, indicating SVAs adopt properties of cis-regulatory elements in the absence of repression. Notably, genome-wide derepression of SVAs elicited the communal up-regulation of KZNFs that reside in KZNF clusters. This phenomenon may provide new insights into the potential mechanisms used by the host genome to sense and counteract TE invasions.

Tyrosine hydroxylase phosphorylation is under the control of serine 40.

Tyrosine hydroxylase catalyzes the initial and rate-limiting step in the biosynthesis of the neurotransmitter dopamine. The phosphorylation state of Ser40 and Ser31 is believed to exert a direct effect on the enzymatic activity of tyrosine hydroxylase. Interestingly, some studies report that Ser31 phosphorylation affects Ser40 phosphorylation, while Ser40 phosphorylation has no effect on Ser31 phosphorylation, a process named hierarchical phosphorylation. Here, we provide a detailed investigation into the signal transduction mechanisms regulating Ser40 and Ser31 phosphorylation in dopaminergic mouse MN9D and Neuro2A cells. We find that cyclic nucleotide signaling drives Ser40 phosphorylation, and that Ser31 phosphorylation is strongly regulated by ERK signaling. Inhibition of ERK1/2 with UO126 or PD98059 reduced Ser31 phosphorylation, but surprisingly had no effect on Ser40 phosphorylation, contradicting a role for Ser31 in the regulation of Ser40. Moreover, to elucidate a possible hierarchical mechanism controlling tyrosine hydroxylase phosphorylation, we introduced tyrosine hydroxylase variants in Neuro2A mouse neuroblastoma cells that mimic either phosphorylated or unphosphorylated serine residues. When we introduced a Ser40Ala tyrosine hydroxylase variant, Ser31 phosphorylation was completely absent. Additionally, neither the tyrosine hydroxylase variant Ser31Asp, nor the variant Ser31Ala had any significant effect on basal Ser40 phosphorylation levels. These results suggest that tyrosine hydroxylase is not controlled by hierarchical phosphorylation in the sense that first Ser31 has to be phosphorylated and subsequently Ser40, but, conversely, that Ser40 phosphorylation is essential for Ser31 phosphorylation. Overall our study suggests that Ser40 is the crucial residue to target so as to modulate tyrosine hydroxylase activity.

Neuronal Dot1l Activity Acts as a Mitochondrial Gene-Repressor Associated with Human Brain Aging via H3K79 Hypermethylation.

Methylation of histone 3 at lysine 79 (H3K79) and its catalyst, a disrupter of telomeric silencing (DOT1l), have been coupled to multiple forms of stress, such as bioenergetic and ER challenges. However, studies on H3K79 methylation and Dot1l in the (aging) brain and neurons are limited. This, together with the increasing evidence of a dynamic neuroepigenome, made us wonder if H3K79 methylation and its activator Dot1l could play important roles in brain aging and associated disorders. In aged humans, we found strong and consistent global hypermethylation of H3K79 in neurons. Specific in dopaminergic neurons, we found a strong increase in H3K79 methylation in lipofucsin positive neurons, which are linked to pathology. In animals, where we conditionally removed Dot1l, we found a rapid loss of H3K79 methylation. As a consequence, we found some decrease in specific dopaminergic genes, and surprisingly, a clear up-regulation of almost all genes belonging to the family of the respiratory chain. These data, in relation to the observed increase in global H3K79 methylation, suggest that there is an inverse relationship between H3K79 methylation and the capacity of energy metabolism in neuronal systems.

Cue and Reward Evoked Dopamine Activity Is Necessary for Maintaining Learned Pavlovian Associations.

Associating natural rewards with predictive environmental cues is crucial for survival. Dopamine (DA) neurons of the ventral tegmental area (VTA) are thought to play a crucial role in this process by encoding reward prediction errors (RPEs) that have been hypothesized to play a role in associative learning. However, it is unclear whether this signal is still necessary after animals have acquired a cue-reward association. In order to investigate this, we trained mice to learn a Pavlovian cue-reward association. After learning, mice show robust anticipatory and consummatory licking behavior. As expected, calcium activity of VTA DA neurons goes up for cue presentation as well as reward delivery. Optogenetic inhibition during the moment of reward delivery disrupts learned behavior, even in the continued presence of reward. This effect is more pronounced over trials and persists on the next training day. Moreover, outside of the task licking behavior and locomotion are unaffected. Similarly to inhibitions during the reward period, we find that inhibiting cue-induced dopamine (DA) signals robustly decreases learned licking behavior, indicating that cue-related DA signals are a potent driver for learned behavior. Overall, we show that inhibition of either of these DA signals directly impairs the expression of learned associative behavior. Thus, continued DA signaling in a learned state is necessary for consolidating Pavlovian associations.SIGNIFICANCE STATEMENT Dopamine (DA) neurons of the ventral tegmental area (VTA) have long been suggested to be necessary for animals to associate environmental cues with rewards that they predict. Here, we use time-locked optogenetic inhibition of these neurons to show that the activity of these neurons is directly necessary for performance on a Pavlovian conditioning task, without affecting locomotor per se These findings provide further support for the direct importance of second-by-second DA neuron activity in associative learning.

Tcf4 is required for correct brain development during embryogenesis.

Tcf4 has been linked to autism, schizophrenia, and Pitt-Hopkins Syndrome (PTHS) in humans, suggesting a role for Tcf4 in brain development and importantly cortical development. However, the mechanisms behind its role in disease and brain development are still elusive. We provide evidence that Tcf4 has a critical function in the differentiation of cortical regions, corpus callosum and anterior commissure formation, and development of the hippocampus during murine embryonic development. In the present study, we show that Tcf4 is expressed throughout the developing brain at the peak of neurogenesis. Deletion of Tcf4 results in mis-specification of the cortical neurons, malformation of the corpus callosum and anterior commissure, and hypoplasia of the hippocampus. Furthermore, the Tcf4 mutant shows an absence of midline remodeling, underlined by the loss of GFAP-expressing midline glia in the indusium griseum and callosal wedge and midline zipper glia in the telencephalic midline. RNA-sequencing on E14.5 cortex material shows that Tcf4 functions as a transcriptional activator and loss of Tcf4 results in downregulation of genes linked to neurogenesis and neuronal maturation. Furthermore, many genes that are differentially expressed after Tcf4 ablation are linked to other neurodevelopmental disorders. Taken together, we show that correct brain development and neuronal differentiation are severely affected in Tcf4 mutants, phenocopying morphological brain defects detected in PTHS patients. The presented data identifies new leads to understand the mechanisms behind brain and specifically cortical development and can provide novel insights in developmental mechanisms underlying human brain defects.

Nkx2.9 Contributes to Mid-Hindbrain Patterning by Regulation of mdDA Neuronal Cell-Fate and Repression of a Hindbrain-Specific Cell-Fate.

Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.

Acquisition of the Midbrain Dopaminergic Neuronal Identity.

The mesodiencephalic dopaminergic (mdDA) group of neurons comprises molecularly distinct subgroups, of which the substantia nigra (SN) and ventral tegmental area (VTA) are the best known, due to the selective degeneration of the SN during Parkinson's disease. However, although significant research has been conducted on the molecular build-up of these subsets, much is still unknown about how these subsets develop and which factors are involved in this process. In this review, we aim to describe the life of an mdDA neuron, from specification in the floor plate to differentiation into the different subsets. All mdDA neurons are born in the mesodiencephalic floor plate under the influence of both SHH-signaling, important for floor plate patterning, and WNT-signaling, involved in establishing the progenitor pool and the start of the specification of mdDA neurons. Furthermore, transcription factors, like Ngn2, Ascl1, Lmx1a, and En1, and epigenetic factors, like Ezh2, are important in the correct specification of dopamine (DA) progenitors. Later during development, mdDA neurons are further subdivided into different molecular subsets by, amongst others, Otx2, involved in the specification of subsets in the VTA, and En1, Pitx3, Lmx1a, and WNT-signaling, involved in the specification of subsets in the SN. Interestingly, factors involved in early specification in the floor plate can serve a dual function and can also be involved in subset specification. Besides the mdDA group of neurons, other systems in the embryo contain different subsets, like the immune system. Interestingly, many factors involved in the development of mdDA neurons are similarly involved in immune system development and vice versa. This indicates that similar mechanisms are used in the development of these systems, and that knowledge about the development of the immune system may hold clues for the factors involved in the development of mdDA neurons, which may be used in culture protocols for cell replacement therapies.

FoxO6 affects Plxna4-mediated neuronal migration during mouse cortical development.

The forkhead transcription factor FoxO6 is prominently expressed during development of the murine neocortex. However, its function in cortical development is as yet unknown. We now demonstrate that cortical development is altered in FoxO6+/- and FoxO6-/- mice, showing migrating neurons halted in the intermediate zone. Using a FoxO6-directed siRNA approach, we substantiate the requirement of FoxO6 for a correct radial migration in the developing neocortex. Subsequent genome-wide transcriptome analysis reveals altered expression of genes involved in cell adhesion, axon guidance, and gliogenesis upon silencing of FoxO6 We then show that FoxO6 binds to DAF-16-binding elements in the Plexin A4 (Plxna4) promoter region and affects Plxna4 expression. Finally, ectopic Plxna4 expression restores radial migration in FoxO6+/- and siRNA-mediated knockdown models. In conclusion, the presented data provide insights into the molecular mechanisms whereby transcriptional programs drive cortical development.

Epigenetic mechanisms in the development and maintenance of dopaminergic neurons.

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral mesodiencephalon and are involved in psychiatric disorders and severely affected in neurodegenerative diseases such as Parkinson's disease. mdDA neuronal development has received much attention in the last 15 years and many transcription factors involved in mdDA specification have been discovered. More recently however, the impact of epigenetic regulation has come into focus, and it's emerging that the processes of histone modification and DNA methylation form the basis of genetic switches that operate during mdDA development. Here, we review the epigenetic control of mdDA development, maturation and maintenance. As we highlight, epigenetic mechanisms play a pivotal role in all of these processes and the knowledge gathered from studying epigenetics in these contexts may aid our understanding of mdDA-related pathologies.

Specification of dopaminergic subsets involves interplay of En1 and Pitx3.

Mesodiencephalic dopaminergic (mdDA) neurons control locomotion and emotion and are affected in multiple psychiatric and neurodegenerative diseases, including Parkinson's disease (PD). The homeodomain transcription factor Pitx3 is pivotal in mdDA neuron development and loss of Pitx3 results in programming deficits in a rostrolateral subpopulation of mdDA neurons destined to form the substantia nigra pars compacta (SNc), reminiscent of the specific cell loss observed in PD. We show here that in adult mice in which the gene encoding a second homeoprotein, engrailed 1 (En1), has been deleted, dramatic loss of mdDA neurons and striatal innervation defects were observed, partially reminiscent of defects observed in Pitx3(-/-) mice. We then continue to reveal developmental crosstalk between En1 and Pitx3 through genome-wide expression analysis. During development, both En1 and Pitx3 are required to induce expression of mdDA genes in the rostrolateral subset destined to form the SNc. By contrast, Pitx3 and En1 reciprocally regulate a separate gene cluster, which includes Cck, demarcating a caudal mdDA subset in wild-type embryos. Whereas En1 is crucial for induction of this caudal phenotype, Pitx3 antagonizes it rostrolaterally. The combinatorial action of En1 and Pitx3 is potentially realized through at least three levels of molecular interaction: (1) influencing each other's expression level, (2) releasing histone deacetylase-mediated repression of Nurr1 target genes and (3) modulating En1 activity through Pitx3-driven activation of En1 modulatory proteins. These findings show how two crucial mediators of mdDA neuronal development, En1 and Pitx3, interact in dopaminergic subset specification, the importance of which is exemplified by the specific vulnerability of the SNc found in PD.

S-nitrosylation of HDAC2 regulates the expression of the chromatin-remodeling factor Brm during radial neuron migration.

Dynamic epigenetic modifications play a key role in mediating the expression of genes required for neuronal development. We previously identified nitric oxide (NO) as a signaling molecule that mediates S-nitrosylation of histone deacetylase 2 (HDAC2) and epigenetic changes in neurons. Here, we show that HDAC2 nitrosylation regulates neuronal radial migration during cortical development. Bead-array analysis performed in the developing cortex revealed that brahma (Brm), a subunit of the ATP-dependent chromatin-remodeling complex BRG/brahma-associated factor, is one of the genes regulated by S-nitrosylation of HDAC2. In the cortex, expression of a mutant form of HDAC2 that cannot be nitrosylated dramatically inhibits Brm expression. Our study identifies NO and HDAC2 nitrosylation as part of a signaling pathway that regulates cortical development and the expression of Brm in neurons.

FoxK2 is required for cellular proliferation and survival.

FoxK2 is a forkhead transcription factor expressed ubiquitously in the developing murine central nervous system. Here we investigated the role of FoxK2 in vitro and focused on proliferation and cellular survival. Knockdown of FoxK2 results in a decrease in BrdU incorporation and H3 phosphorylation, suggesting attenuation of proliferation. In the absence of growth factors, FoxK2 knockdown results in a dramatic increase in caspase 3 activity and propidium iodide positive cells, indicative of cell death. Additionally, knockdown of FoxK2 results in an increase in the mRNA of Gadd45α, Gadd45γ, as well as an increase in the phosphorylation of the mTOR dependent kinase p70S6K. Rapamycin treatment completely blocked the increase in p70S6K and synergistically potentiated the decrease in H3 phosphorylation upon FoxK2 knockdown. To gain more insight into the proapoptotic effects upon FoxK2 knockdown we screened for changes in Bcl2 genes. Upon FoxK2 knockdown both Puma and Noxa were significantly upregulated. Both genes were not inhibited by rapamycin treatment, instead rapamycin increased Noxa mRNA. FoxK2 requirement in cellular survival is further emphasized by the fact that resistance to TGFß-induced cell death was greatly diminished after FoxK2 knockdown. Overall our data suggest FoxK2 is required for proliferation and survival, that mTOR is part of a feedback loop partly compensating for FoxK2 loss, possibly by upregulating Gadd45s, whereas cell death upon FoxK2 loss is induced in a Bcl2 dependent manner via Puma and Noxa.

Molecular mechanisms of dopaminergic subset specification: fundamental aspects and clinical perspectives.

Dopaminergic (DA) neurons in the ventral mesodiencephalon control locomotion and emotion and are affected in psychiatric and neurodegenerative diseases, such as Parkinson's disease (PD). A clinical hallmark of PD is the specific degeneration of DA neurons located within the substantia nigra (SNc), whereas neurons in the ventral tegmental area remain unaffected. Recent advances have highlighted that the selective vulnerability of the SNc may originate in subset-specific molecular programming during DA neuron development, and significantly increased our understanding of the molecular code that drives specific SNc development. We here present an up-to-date overview of molecular mechanisms that direct DA subset specification, integrating our current knowledge about subset-specific roles of transcription factors, signaling pathways and morphogenes. We discuss strategies to further unravel subset-specific gene-regulatory networks, and the clinical promise of fundamental knowledge about subset specification of DA neurons, with regards to cell replacement therapy and cell-type-specific vulnerability in PD.

Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons.

Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is displayed only by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we have demonstrated that Pitx3(-/-) embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA signaling in Pitx3(-/-) embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In the search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and Th are regulated by Pitx3 and RA signaling, which influences the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA signaling represents only one aspect of the Pitx3 downstream cascade, as Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade, suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.

Distinct temporal expression of 5-HT(1A) and 5-HT(2A) receptors on cerebellar granule cells in mice.

Serotonin plays an important role of controlling the physiology of the cerebellum. However, serotonin receptor expression has not been fully studied in the developing cerebellum. We have recently shown that cerebellar granule cells transiently express 5-HT3 receptors. In the present study, we investigate expression of 5-HT1 and 5-HT2 receptors in the mouse cerebellum both during postnatal development and in juvenile mice. Here, we show for the first time that 5-HT1A and 5-HT2A receptors are present on cerebellar granule cells with a distinct temporal expression pattern: 5-HT1A receptors are expressed only during the first 2 weeks, while 5-HT2A receptor expression persists until at least 8 weeks after birth. Because of its prolonged expression pattern, we investigated the electrophysiological properties of the 5-HT2A receptor. 5-HT2A receptors expressed by cerebellar granule cells promote stability by reducing variability of the synaptic response, and they modulate the paired-pulse ratio of the parallel fibre-Purkinje cell synapse. Furthermore, pharmacological block of 5-HT2A receptors enhances short-term synaptic plasticity at the parallel fibre-Purkinje cell synapse. We thus show a novel role for serotonin in controlling function of the cerebellum via 5-HT2A receptors expressed by cerebellar granule cells.

Boosting endogenous dopamine production: a novel therapeutic approach for Parkinson's disease.

Innovative therapeutic strategies are urgently needed for Parkinson's disease due to limited efficacy of current treatments and a weak therapeutic pipeline. In this forum article, we propose targeting tyrosine hydroxylase phosphorylation as a novel mechanism of action to address this critical need.

Morphine withdrawal enhances constitutive µ-opioid receptor activity in the ventral tegmental area.

µ-Opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive activity is highly relevant in the mouse VTA, as it regulates GABAergic input to dopamine neurons. Specifically, suppression of MOR constitutive activity with the inverse agonist KC-2-009 enhanced GABAergic neurotransmission onto VTA dopamine neurons. This inverse agonistic effect was fully blocked by the specific MOR neutral antagonist CTOP, which had no effect on GABAergic transmission itself. We next show that withdrawal from chronic morphine further increases the magnitude of inverse agonistic effects at the MOR, suggesting enhanced MOR constitutive activity. We demonstrate that this increase can be an adaptive response to the detrimental elevation in cAMP levels known to occur during morphine withdrawal. These findings offer important insights in the physiological occurrence and function of MOR constitutive activity, and have important implications for therapeutic strategies aimed at normalizing MOR signaling during addiction and opioid overdose.

Lmx1a is an activator of Rgs4 and Grb10 and is responsible for the correct specification of rostral and medial mdDA neurons.

The LIM homeodomain transcription factor Lmx1a is a very potent inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyse the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A-overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other previously generated expression arrays and on their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (dr/dr) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a-mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A-overexpression cell system resulted in the identification of novel direct or indirect downstream targets of Lmx1a in mdDA neurons: Grb10, Rgs4 and Vmat2.

Tcf4 Is Involved in Subset Specification of Mesodiencephalic Dopaminergic Neurons.

During development, mesodiencephalic dopaminergic (mdDA) neurons form into different molecular subsets. Knowledge of which factors contribute to the specification of these subsets is currently insufficient. In this study, we examined the role of Tcf4, a member of the E-box protein family, in mdDA neuronal development and subset specification. We show that Tcf4 is expressed throughout development, but is no longer detected in adult midbrain. Deletion of Tcf4 results in an initial increase in TH-expressing neurons at E11.5, but this normalizes at later embryonic stages. However, the caudal subset marker Nxph3 and rostral subset marker Ahd2 are affected at E14.5, indicating that Tcf4 is involved in correct differentiation of mdDA neuronal subsets. At P0, expression of these markers partially recovers, whereas expression of Th transcript and TH protein appears to be affected in lateral parts of the mdDA neuronal population. The initial increase in TH-expressing cells and delay in subset specification could be due to the increase in expression of the bHLH factor Ascl1, known for its role in mdDA neuronal differentiation, upon loss of Tcf4. Taken together, our data identified a minor role for Tcf4 in mdDA neuronal development and subset specification.

Advancing urban mental health research: from complexity science to actionable targets for intervention.

Urbanisation and common mental disorders (CMDs; ie, depressive, anxiety, and substance use disorders) are increasing worldwide. In this Review, we discuss how urbanicity and risk of CMDs relate to each other and call for a complexity science approach to advance understanding of this interrelationship. We did an ecological analysis using data on urbanicity and CMD burden in 191 countries. We found a positive, non-linear relationship with a higher CMD prevalence in more urbanised countries, particularly for anxiety disorders. We also did a review of meta-analytic studies on the association between urban factors and CMD risk. We identified factors relating to the ambient, physical, and social urban environment and showed differences per diagnosis of CMDs. We argue that factors in the urban environment are likely to operate as a complex system and interact with each other and with individual city inhabitants (including their psychological and neurobiological characteristics) to shape mental health in an urban context. These interactions operate on various timescales and show feedback loop mechanisms, rendering system behaviour characterised by non-linearity that is hard to predict over time. We present a conceptual framework for future urban mental health research that uses a complexity science approach. We conclude by discussing how complexity science methodology (eg, network analyses, system-dynamic modelling, and agent-based modelling) could enable identification of actionable targets for treatment and policy, aimed at decreasing CMD burdens in an urban context.